Substitution of His59 converts CD39 apyrase into an ADPase in a quaternary structure dependent manner

The two transmembrane domains of CD39 ecto-apyrase regulate the formation of fully active homotetramers. We show that mutations in apyrase conserved region 1 (ACR1) have two dramatically different sets of effects determined by whether they occur in intact tetramers or in disrupted tetramers or monomers. In intact tetramers, substitution of H59 in the rat brain CD39 ACR1 with G or S abolishes more than 90% of the ATPase activity but less than 50% of the ADPase activity, converting the enzyme into an ADPase with relative ADP:ATP hydrolysis rates of 6:1 or 8:1, respectively. In contrast, the same substitutions in tetramers lacking either transmembrane domain, in monomers lacking both transmembrane domains, or in detergent-solubilized full-length monomers have no effect on ATPase activity and increase ADPase activity approximately 2-fold, resulting in equal ATPase and ADPase activities. N61R substitution has a much smaller effect on the ADPase:ATPase ratio in both cases. While the data for truncated and monomeric constructs are consistent with the proposed role of ACR1 as the beta-phosphate binding domain by analogy with the actin/hsp70/hexokinase superfamily, the finding that H59 substitutions in full-length CD39 primarily diminish the ATP hydrolysis rate suggests that ACR1 may play a different role in intact tetramers. We propose that CD39 uses different ATPase and ADPase mechanisms in different quaternary structure contexts, and that H59 in ACR1 plays a central role specifically in ATP hydrolysis in intact tetramers.     

Studies on the nature of adenosine diphosphatase activity from rat liver mitochondria

Adenosine diphosphatase (ADPase) activity was studied in rat liver with [beta-32P]ADP as a substrate. Mitochondria and outer mitochondrial membrane fractions were isolated and assayed for ADPase and various marker enzymes. ADPase activity was strikingly reduced when the outer membranes were removed from the mitochondria whether by digitonin treatment or osmotic shock. Addition of the inter-membrane space subfraction to the purified outer membranes resulted in enhanced ADPase activity. Addition of the inter-mitochondrial membrane enzyme adenylate kinase to outer membranes also produced a large stimulation of activity. The ADPase activity could also be reconstituted in vitro with adenylate kinase and either mitoplast ATPase or ouabain-sensitive (Na+ + K+ + Mg2+)-ATPase. Chloroform-released ATPase, however, was not capable of producing an ADPase activity when combined with adenylate kinase. Gel permeation chromatography of Triton-solubilised outer mitochondrial membranes was unable to resolve ADPase activity from contaminating ATPase. These results suggest that the majority of ADPase activity in rat liver mitochondria consists of the coupled activity of adenylate kinase and ATPase.     

Transmembrane domains confer different substrate specificities and adenosine diphosphate hydrolysis mechanisms on CD39, CD39L1, and chimeras

Members of the ecto-nucleoside triphosphate diphosphohydrolase (eNTPDase) family exhibit distinctive substrate specificities, but how such specificities are achieved by enzymes with identical putative catalytic domains is unknown. Previously we showed that H59G substitution changes CD39 from an apyrase to an adenosine diphosphatase (ADPase) in a manner that depends on intact associations of both transmembrane domains with the membrane. Here we show that the extracellular domain of CD39L1 ecto-adenosine triphosphatase (ecto-ATPase) has the same 3:1 ATP:ADP hydrolysis ratio as the extracellular domain of CD39, suggesting that the transmembrane domains are required to confer the native substrate specificities on each enzyme. As in CD39, H50G substitution has little effect on the activity of the CD39L1 extracellular domain or solubilized monomers. However, H50G substitution diminishes both ATPase and ADPase activities of native CD39L1, in contrast to its selective effect on ATPase activity in CD39, suggesting that the transmembrane domains confer different ADP hydrolysis mechanisms on CD39 and CD39L1. We then show that the transmembrane domains of CD39L1 can substitute for those of CD39 in conferring native CD39 substrate specificity and regulation of H59 but that the transmembrane domains of CD39 confer neither CD39 nor CD39L1 properties on the CD39L1 extracellular domain. These results suggest that non-apyrase conserved region residues in the extracellular domain contain the information specifying CD39 native properties but have a nonspecific requirement for two transmembrane domains to manifest the information.     

Subcellular localization and properties of adenosine diphosphatase activity in human polymorphonuclear leukocytes

Adenosine diphosphatase (ADPase) activities were studied in human polymorphonuclear leukocytes with a recently developed radio-assay. The neutrophils were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation. The sucrose density gradient fractions were assayed for ADPase activity and for principal organelle marker enzymes. ADPase activity was distributed between the plasma membrane, specific granule and soluble fractions. The plasma membrane and specific granule activities had similar kinetic and inhibitor properties but the cytosolic enzyme was clearly different. Studies with the non-penetrating inhibitor diazotized sulphanilic acid and measurements of latent activity indicate that plasma membrane ADPase activity is located on the external aspect to the cell. Its possible role in inhibiting platelet aggregation is discussed. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (mU/mg protein) of ADPase activity, in contrast to those of alkaline phosphatase, were similar in all three groups. This result, together with fractionation experiments and inhibition studies strongly suggests that ADPase activity is not attributable to neutrophil alkaline phosphatase.     

Demonstration of plasma-membrane adenosine diphosphatase activity in rat lung

The adenosine diphosphatase (ADPase) activity of rat lung has been investigated. Subcellular fractionation of lung tissue homogenates by sucrose density gradient centrifugation has shown the ADPase activity to be associated with the plasma membrane. ADPase was solubilised from the membranes and fractionated by ammonium sulphate precipitation to separate a specific, low-Km ADPase from non-specific alkaline phosphatase activity. The solubilised ADPase has a Km of 50 microM at pH 7.5 and appears to be distinct from ATPase.     

Tissue distribution of adenosine diphosphatase activity in the rat

The specific activity of adenosine diphosphatase (ADPase) was determined in rat tissue homogenates using a specific radioassay. 14 different tissues were sampled and activity was found in most homogenates examined. The levels ranged from very little activity in the pancreas to high levels in the duodenum and ileum. High levels of ADPase activity found in the gastrointestinal tract could be partially attributed to non-specific alkaline phosphatase. It is concluded that ADPase activity is widely distributed in rat tissues.     

Subcellular localization and properties of rat liver adenosine diphosphatase.

ADPase (adenosine diphosphatase) was assayed in rat liver homogenates with [beta-32P]ADP as substrate. The activity had a pH optimum of 8.0 and was strongly activated by Mg2+. The intracellular localization was determined by analytical subcellular fractionation with single-step sucrose-density-gradient centrifugation. Selective membrane perturbants were used to enhance the resolution of the various organelles. ADPase was localized to the mitochondria. Mitochondria were isolated by differential centrifugation and subfractionated by selective disruption of the inner and outer membranes. The intramitochondrial localization of ADPase was compared with various marker enzymes and was shown to be concentrated in the outer-membrane fractions. The effects of various inhibitors on the ADPase activity were determined and the possibility that the activity could be due to known enzyme systems was considered. It is concluded that ADP degradation is due to a hitherto unrecognized mitochondrial enzyme.     

Effects of protein-modifying reagents on an isoenzyme of potato apyrase

Treatment of an isoenzyme of potato apyrase of high adenosine triphosphatase/adenosine diphosphatase (ATPase/ADPase) ratio with iodine, N-acetylimidazole or tetranitromethane inactivates the ATPase activity of this enzyme faster than its ADPase activity. There was protection by substrates with the two last-named substances. This and the appearance of nitrotyrosine suggests the participation of tyrosyl residues in both enzymic activities of potato apyrase. The participation of thiol groups is excluded by the insensitivity of apyrase to p-chloromercuribenzoate. Also, 2-hydroxy-5-nitrobenzyl bromide or carboxymethylation produce the same rate of inactivation of ATPase and ADPase activities. Substrates protect both activities from inactivation. Hydrogen peroxide and photo-oxidation inactivate ATPase activity faster than ADPase activity. There is no protection by substrates. Analysis of pH effects on V_max. and K_m suggest different pK values for the amino acid residues at the ATP and ADP sites.     

Properties and subcellular localization of adenosine diphosphatase in arterial smooth muscle cells in culture

The properties and subcellular localization of adenosine diphosphatase (ADPase) activity in smooth muscle cells cultured from pig aortas have been investigated. The pH optimum of ADPase activity was 7.3 and the apparent K_m for ADP was 10.3 μM. ADPase activity was inhibited completely by EDTA and was restored by the addition of divalent cations. The enzyme activity was not inhibited by 2-glycerophosphate, a substrate for non-specific phosphatases, nor by levamisole, a specific inhibitor of alkaline phosphatase. Smooth muscle cells were homogenized and a post-nuclear supernatant was applied to a sucrose density gradient in a Beaufay automatic zonal rotor. The distribution of ADPase activity in the density gradient was similar to that of 5′-nucleotidase activity, a marker enzyme for the plasma membrane, and distinct from the distributions of the marker enzymes for the other organelles. When the cells were homogenized in the presence of digitonin, an agent which binds to cholesterol and increases the equilibrium density of the plasma membrane, the modal equilibrium densities of ADPase activity and of 5′-nucleotidase activity were increased to similar extents, thus confirming the plasma membrane localization of ADPase activity.     

Properties and subcellular localization of adenosine diphosphatase in rat heart

Some properties and subcellular localization of adenosine diphosphatase (ADPase) activity from rat heart have been investigated. The pH optimum was 7.4, maximal activity was found with 5 mM MgCl2, and the apparent Km was 20 microM. ADPase activity was strongly inhibited by NaF and AppNHp, and to a lesser extent by AMP and GppNHp. The enzyme was not inhibited by p-nitrophenylphosphate, beta-glycerophosphate, or pyridoxal phosphate. The distribution of ADPase activity in subcellular fractions obtained by differential centrifugation parallel ouabain-sensitive (Na+-K+)ATPase and 5'-nucleotidase activities, suggesting a plasma membrane-bound localization. The functional significance of ADPase in adenosine production and hemostasis is discussed.     

Small‐molecule reductants inhibit multicatalytic activity of AA‐NADase from Agkistrodon acutus venom by reducing the disulfide‐bonds and Cu(II) of enzyme

AA‐NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA‐NADase contains Cu(II) and has disulfide‐bond linkages between two peptide chains. The effects of the reduction of the disulfide‐bonds and Cu(II) in AA‐NADase by small‐molecule reductants on its NADase and ADPase activities have been investigated by polyacrylamide gel electrophoresis, high performance liquid chromatography, electron paramagnetic resonance spectroscopy and isothermal titration calorimetry. The results show that AA‐NADase has six disulfide‐bonds and fifteen free cysteine residues. L‐ascorbate inhibits AA‐NADase on both NADase and ADPase activities through the reduction of Cu(II) in AA‐NADase to Cu(I), while other reductants, dithiothreitol, glutathione and tris(2‐carboxyethyl)phosphine inhibit both NADase and ADPase activities through the reduction of Cu(II) to Cu(I) and the cleavage of disulfide‐bonds in AA‐NADase. Apo‐AA‐NADase can recover its NADase and ADPase activities in the presence of 1 mM Zn(II). However, apo‐AA‐NADase does not recover any NADase or ADPase activity in the presence of 1 mM Zn(II) and 2 mM TCEP. The multicatalytic activity relies on both disulfide‐bonds and Cu(II), while Cu(I) can not activate the enzyme activities. AA‐NADase is probably only active as a dimer. The inhibition curves for both ADPase and NADase activities by each reductant share a similar trend, suggesting both ADPase and NADase activities probably occur at the same site. In addition, we also find that glutathione and L‐ascorbate are endogenous inhibitors to the multicatalytic activity of AA‐NADase. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 141–149, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Phospholipase C axis is the preferential pathway leading to PKC activation following PTH or PTHrP stimulation in human term placenta

In the present report we describe an NTPDase 1 (ATP diphosphohydrolase; ecto-apyrase; EC 3.6.1.5) in rat hippocampal slices. The effect of glutamate on the ATPase and ADPase activities in rat hippocampal slices of different ages was also studied since adenosine, the final product of an enzymatic chain that includes NTPDase 1 and 5'-nucleotidase, can act upon A1 receptors in turn decreasing the release of glutamate. Hippocampal slices from 7, 14, 20-23 and 60 day-old rats were prepared and ATPase and ADPase activities were measured. The parallelism of ATPase and ADPase activities in all parameters tested indicated the presence of an ATP diphosphohydrolase. In addition, a Chevillard plot indicated that ATP and ADP are hydrolyzed at the same active site on the enzyme. ATPase activity was significantly activated by glutamate in 20-23 and 60 day-old rats, but ADPase activity was not activated. These results could indicate distinct behavior of the ATPase and ADPase activities of NTPDase 1 in relation to glutamate or the simultaneous action of the ecto-ATPase. Activation of ATPase activity by glutamate may constitute an important role in this developmental period, possibly protecting against the neurotoxicity induced by ATP, as well as producing high levels of ADP, by increasing adenosine production, a neuroprotective compound.     

Distribution of ADPase activity in the lactating rat mammary gland and its possible role in an ATP cycle in the Golgi apparatus.

A Ca2+/Mg(2+)-stimulated ADPase has been found to occur in the lactating rat mammary gland. The enzyme is membrane associated and occurs in mitochondrial, microsomal, and Golgi apparatus fractions. The pH activity curves for the Golgi apparatus and microsomal fractions display two distinct maxima, one at pH 6.3 and one at pH 7.4. Studies with inhibitors and activators indicate that the enzyme is similar to ADPases found in other tissues and is distinct from the uridine nucleoside diphosphatase previously reported in the mammary Golgi apparatus. The occurrence of ADPase in the Golgi apparatus indicates a possible role for this enzyme in the milk secretory process, while the microsomal enzyme could be involved in extracellular activities.     

Characterization and immunohistochemical localization of nucleoside triphosphate diphosphohydrolase (NTPDase) in pig adrenal glands (presence of a non-sedimentable isoform)

Considering that adrenal glands possess a variety of purinoceptors associated with various cell types and that some of these cells (chromaffin cells) secrete large amounts of adenine nucleotides, it was of interest to localize nucleoside triphosphate diphosphohydrolase (NTPDase) in these glands and to define the biochemical characteristics of this ectonucleotidase. Immunolocalization produced a moderate reaction in capsula and medulla, with no signal in zona glomerulosa and zona reticularis. In contrast, a very strong reaction was found in zona fasciculata. Biochemical analysis of particulate fractions isolated from whole glands revealed high levels of ATPase and ADPase activities. This appeared to be attributable to the NTPDase since the level of ADPase was as high as ATPase. Both ATPase and ADPase activities were similarly inhibited by sodium azide. Additionally electrophoretograms with these two substrates showed comparable patterns. Western blots with 'Ringo', an antibody that recognizes the different isoforms of mammalian NTPDases, showed the presence of isoforms of NTPDases at 54 and 78 kDa, respectively. Interestingly, the 54 kDa isoform remains in the supernatant of a chromaffin granule lysate after ultracentrifugation. Up until now little interest has been given to the relationship between adrenal medulla and cortex. Presence of purinoceptors and ectonucleotidases in both these regions together with the effects of ATP in vivo and in vitro in different species indicate that purines play a significant role in adrenal glands.     

Lysosomal membrane adenosine triphosphatase; solubilization and partial characterization

Membranes prepared from Triton WR-1339-filled lysosomes (tritosomes) contained ATPase activity with a pH optimum of 5–8. These membranes also showed adenosine diphosphatase, adenosine monophosphatase, acid β-glycerol phosphatase, and acid pyrophosphatase activities. The soluble (nonmembrane) fraction of the tritosomes also contained these activities, but the properties of the soluble adenine nucleotide phosphatase activities were different from the membrane-associated enzymes. The pH optimum of tritosomal membrane ATPase changed to 5 after solubilization with Triton X-100, but ADPase and AMPase optima remained at 6–7. The pH optimum of intact membrane ATPase was also 5 when the substrate was α,β-methylene-ATP. Thus, tritosomal membrane ATPase apparently exhibits a pH 8 optimum only when acting in concert with ADPase and AMPase in intact membranes. Rates of ATP hydrolysis to adenosine were also significantly greater in intact membranes than in Triton X-100-solubilized fraction. Centrifugation of Triton X-100-solubilized tritosomal membranes in sucrose density gradients showed that ATPase and ADPase activities sedimented to one peak, and that AMPase, acid phosphatase, and pyrophosphatase were grouped in another peak. Thus, tritosomal membrane ATPase activity was not due to the latter enzymes. The resulting purification was about fourfold for ATPase. The Mr for ATPase and ADPase was estimated to be about 65,000 and for AMPase, acid phosphatase, and pyrophosphatase about 200,000.     

Differential effects of magnesium on the hydrolysis of ADP and ATP in human term placenta. Effect of substrates and potassium

This study evaluates the effect of Mg2+ on the extramitochondrial hydrolysis of ATP and ADP by human term placental mitochondria (HPM) and submitochondrial particle (SMP). Extramitochondrial ATPase and ADPase activities were evaluated in the presence or absence of K+, and different oxidizable substrates. Mg2+ increased both ATP and ADP hydrolysis according to the experimental conditions, and this stimulation was related to the mitochondrial intactness. The ADPase activity in intact mitochondria is 100-fold higher in presence of K+, succinate and 1mM Mg2+ while this activity is only increased by two-fold on the SMP when compared to the sample without Mg2+. It is clearly demonstrated that up-regulation of these enzyme activities occur in intact mitochondria and not on the enzyme itself. The results suggest that the regulation of ATP and ADP hydrolysis is complex, and Mg2+ plays an important role in the modulation of the extramitochondrial ATPase and ADPase activities in HPM     

Site-directed mutagenesis of human endothelial cell ecto-ADPase/soluble CD39: requirement of glutamate 174 and serine 218 for enzyme activity and inhibition of platelet recruitment

Endothelial cell CD39/ecto-ADPase plays a major role in vascular homeostasis. It rapidly metabolizes ADP released from stimulated platelets, thereby preventing further platelet activation and recruitment. We recently developed a recombinant, soluble form of human CD39, solCD39, with enzymatic and biological properties identical to CD39. To identify amino acids essential for enzymatic/biological activity, we performed site-directed mutagenesis within the four highly conserved apyrase regions of solCD39. Mutation of glutamate 174 to alanine (E174A) and serine 218 to alanine (S218A) resulted in complete and approximately 90% loss of solCD39 enzymatic activity, respectively. Furthermore, compared to wild-type, S57A exhibited a 2-fold increase in ADPase activity without change in ATPase activity, while the tyrosine 127 to alanine (Y127A) mutant lost 50-60% of both ADPase and ATPase activity. The ADPase activity of wild-type solCD39 and each mutant, except for R135A, was greater with calcium as the required divalent cation than with magnesium, but for ATPase activity generally no such preference was observed. Y127A demonstrated the highest calcium/magnesium ADPase activity ratio, 2.8-fold higher than that of wild-type, even though its enzyme activity was greatly reduced. SolCD39 mutants were further characterized by correlating enzymatic with biological activity in an in vitro platelet aggregation system. Each solCD39 mutant was similar to wild-type in reversing platelet aggregation, except for E174A and S218A. E174A, completely devoid of enzymatic activity, failed to inhibit platelet responsiveness, as anticipated. S218A, with 91% loss of ADPase activity, could still reverse platelet aggregation, albeit much less effectively than wild-type solCD39. Thus, glutamate 174 and serine 218 are essential for both the enzymatic and biological activity of solCD39.     

A novel ATP/ADP hydrolysis activity of hyperthermostable group II chaperonin in the presence of cobalt or manganese ion

A novel ATPase activity that was strongly activated in the presence of either cobalt or manganese ion was discovered in the chaperonin from hyperthermophilic Pyrococcus furiosus (Pfu-cpn). Surprisingly, a significant ADPase activity was also detected under the same conditions. A more extensive search revealed similar nucleotide hydrolysis activities in other thermostable chaperonins. Chaperonin activity, i.e., thermal stabilization and refolding of malate dehydrogenase from the guanidine-hydrochloride unfolded state were also detected for Pfu-cpn under the same conditions. We propose that the novel cobalt/manganese-dependent ATP/ADPase activity may be a common trait of various thermostable chaperonins.     

Metal ions binding to NAD-glycohydrolase from the venom of Agkistrodon acutus: regulation of multicatalytic activity

AA-NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA-NADase contains Cu(2+) ions that are essential for its multicatalytic activity. In this study, the interactions between divalent metal ions and AA-NADase and the effects of metal ions on its structure and activity have been investigated by equilibrium dialysis, isothermal titration calorimetry, fluorescence, circular dichroism, dynamic light scattering and HPLC. The results show that AA-NADase has two classes of Cu(2+) binding sites, one activator site with high affinity and approximately six inhibitor sites with low affinity. Cu(2+) ions function as a switch for its NADase activity. In addition, AA-NADase has one Mn(2+) binding site, one Zn(2+) binding site, one strong and two weak Co(2+) binding sites, and two strong and six weak Ni(2+) binding sites. Metal ion binding affinities follow the trend Cu(2+) > Ni(2+) > Mn(2+) > Co(2+) > Zn(2+), which accounts for the existence of one Cu(2+) in the purified AA-NADase. Both NADase and ADPase activities of AA-NADase do not have an absolute requirement for Cu(2+), and all tested metal ions activate its NADase and ADPase activities and the activation capacity follows the trend Zn(2+) > Mn(2+) > Cu(2+) ~Co(2+) > Ni(2+). Metal ions serve as regulators for its multicatalytic activity. Although all tested metal ions have no obvious effects on the global structure of AA-NADase, Cu(2+)- and Zn(2+)-induced conformational changes around some Trp residues have been observed. Interestingly, each tested metal ion has a very similar activation of both NADase and ADPase activities, suggesting that the two different activities probably occur at the same site.     

Characterization and importance of the dimer interface of human calcium-activated nucleotidase

Human calcium-activated nucleotidase (CAN) exists as both a membrane-bound form in the endoplasmic reticulum and pre-Golgi intermediate membranes and as a secreted, soluble form. Although the wild-type human enzyme hydrolyzes ADP poorly, engineered soluble human proteins (SCANs) hydrolyze ADP much more efficiently, making them potentially useful therapeutic proteins for treatment of human clotting pathologies. According to the crystal structure and the recently identified dimeric nature of the soluble nucleotidase, the dimer interface contains a central core of hydrophobic residues. Previously, we demonstrated that the mutation of glutamic acid 130 (located in the dimer interface) to tyrosine increased both the tendency to form dimers and the ADPase activity. In the present study, we investigated the importance of the dimeric state for enzymatic activity and biological function in this nucleotidase by mutating isoleucine 170, which is located in the center of the hydrophobic core of the dimer interface. The results of analytical ultracentrifugation, chemical cross-linking, and tryptophan fluorescence analyses demonstrated that mutation of isoleucine 170 to either positively or negatively charged amino acids (lys or glu) disrupted the calcium-dependent dimerization in soluble CAN. Furthermore, these mutations decreased maximal ADPase activity for both the soluble and membrane-bound enzymes. Although not as critical as the hydrophobic interactions centered at isoleucine 170, the role of hydrophilic interactions in dimer formation was also demonstrated. Thus, mutation of aspartic acid 228 to threonine (D228T) decreased both the tendency to form dimers and ADPase activity, while double mutation of D228T/K224N largely restored the ability to form dimers and the ADPase activity, further indicating that the nucleotidase activity of CAN is linked to its quaternary structure. Since ADPase activity of the soluble form is crucial for its potential development as a therapeutic protein, these findings have implications for engineering the soluble human calcium-activated nucleotidase for clinical applications. In addition, future comparison of monomeric (I170K and I170E mutants) and dimeric (wild-type) crystal structures of SCAN will advance our understanding of its enzymatic mechanism and aid in engineering efforts.